The classical geneticist works by creating phenotype, and then explaining it. A phenotype is one of two or more “alternative states” of a phenomenon. The phenomenon might be very familiar (having five fingers on each hand, or having two eyes). Or it might be very complicated (being able to think; regulating one’s blood sugar within narrow limits). Where most people have five fingers on each hand, some might have only one finger on each hand. This would be a phenotype, and when explained, it might tell us how and why five fingers normally form. In the modern era, geneticists have been able to explain phenotype with amazing speed once they are created.

Some geneticists work with humans; some with mice; some with worms; some with fruit flies; some with plants. All attempt to create and solve phenotypes. Otherwise they are not geneticists in the classical sense of the word!

Why do geneticists start with phenotype? What advantages does this hold? [back]

All science begins with phenomena. Most scientists examine phenomena using the scientific method. They form hypotheses and perform experiments to test their validity. Geneticists, alone among scientists, are capable of taking a different approach. Rather than forming hypotheses, they create phenotypes: alternative states of the phenomenon that interests them. They do this secure in the knowledge that they can determine the causes of phenotypes by positional cloning, and thus establish fundamental requirements for the phenomenon.

The classical genetic approach is extremely powerful because:

It reveals phenomena of which biologists were previously unaware (sometimes because phenomena hide in plain sight).

It permits all proteins with non-redundant function in a particular phenomenon to be discovered.

It is unbiased.

This third point is critically important. Hypothesis-driven research may be influenced by conscious or unconscious wish that a hypothesis will turn out to be correct. The question does not arise in genetics, because there is a tangible and verifiable goal: a phenotype to ascribe to a mutation rather than an hypothesis to test.

ENU (N-ethyl-N-nitrosourea) is the premiere tool for the creation of phenotype in mice.

As just mentioned, phenotype (phenovariance) is the basis of all work in classical genetics. ENU is a powerful alkylating agent that chemically modifies DNA in somatic cells and in cells of the germline. It is generally regarded as a near-perfect germline mutagen for genetic studies in mice. When male mice are treated with ENU, approximately 3,000 mutations (mostly A→T transversions or A→G transitions) are induced at random across the haploid genome (in other words, about one base pair per million is altered). About 1.5% of these mutations fall within coding regions, and about 75% of those alter coding sense. ENU is strictly a point mutagen, and is not known to cause large deletions, inversions, or translocations.

ENU causes transient sterility when administered to male mice owing to attrition of spermatogonia, the stem cells that give rise to sperm. The testis is ultimately repopulated by descendants of a spermatogonial pool that numbers between 10 and 100 cells. Therefore, a single ENU-treated male is usually used to produce only about 20 offspring. If more offspring are examined, there is a good likelihood that the same mutations will be examined over and over again.

ENU-induced mutations, produced in “G0” (Generation 0) males, are transmitted to G1 offspring in a heterozygous state, and often produce dominant phenotype. One of a number of protocols for inbreeding are applied to achieve homozygosity for mutations in the G3 population (Figure 1). These mice can be screened for recessive phenotypes.

The “reverse genetic” approach to gene function will ultimately lead to the targeting of all genes, or their destruction by gene traps. It is likely that conditional knockouts of all genes will one day be available. But this will not end the need for mutagenesis, because in many cases, no visible phenotype results from a knockout. This is not to say that the gene does nothing. Rather, it implies that the investigator does not know what to look for. By starting with phenotype, one may expect to find all genes that have non-redundant function in a given biological process. Moreover, many ENU-induced mutations reveal pleiotropy: many different functions for a given gene. It is commonly observed, for example, that homozygous deletion of a particular gene is lethal, whereas ENU-induced mutations may yield viable variant alleles, sometimes with different visible phenotypic effects.

Causative mutations are identified by the processes of genetic mapping and DNA sequencing. The principle behind genetic mapping is to mix in genetic material that is known not to contribute to the mutant phenotype by outcrossing the mutant stock to another mouse strain (the mapping strain); in progeny that retain the mutant phenotype, loci with alleles contributed by the mapping strain are excluded as causative for the phenotype. By further breeding, the mutation can ultimately be mapped to a small genetic interval and then identified by DNA sequencing. Mutations can now also be identified using whole genome sequencing (please see below).

The advent of next generation sequencing platforms such as the ABI SOLiDTM has lowered the cost and shortened the time required for large-scale sequencing, making the repeated sequencing of whole, individual genomes possible. The genome of any ENU-mutagenized mouse can thus be sequenced and analyzed in entirety within a few weeks. Next generation whole genome sequencing allows the rapid discovery of phenotype-causing mutations. In addition, whole genome sequencing usually reveals a number of mutations that are not responsible for the phenotype of interest (incidental mutations), but potentially deleterious nonetheless. Through identification of such mutations, whole genome sequencing of mutagenized mice may therefore be used to establish an allelic series encompassing all loci.

Incidental mutations are mutations that alter or potentially alter coding sense or transcript splicing, but do not contribute to a particular mutant phenotype. These mutations are found incidentally by whole genome sequencing in the process of looking for mutations that cause a particular phenotype. Many mutations may be identified by whole genome sequencing in a mouse mutant, but generally only one of these mutations will cause the phenotype of interest.

How do you know for sure that a mutation is really the “right” one? [back]

Only about 1 bp per million is altered in the haploid genome of a germ cell in an ENU-treated mouse, and virtually all phenotype emanates from changes in coding sense (nonsense, missense, and splicing mutations). Coding sequence accounts for only about 1.3% of all genomic DNA. Therefore, once a mutation has been mapped to a circumscribed fraction of the genome (the critical region), 1 to 2 million base pairs in length, it is very rare to find more than one coding change within it. Even in substantially larger critical regions, when a mutation is found, it is usually responsible for the phenotype observed. However, added evidence of cause and effect can be adduced by transfection, transgenesis, or gene targeting.

Since each ENU-mutagenized mouse will have a number of potentially deleterious mutations, identification of phenotype-causing mutations requires light mapping to chromosomes. Alternatively and more rapidly, concordance of phenotype with genotype can be established using a large pedigree and used to support causation.

Isn’t a knockout mutation more “sure” to establish the function of a particular gene? [back]

No; it is less sure to do so. Knockouts may produce effects on neighboring genes, and there is no guarantee that when a knockout is produced, it is the sole mutation in the mouse that results. Other mutations may accumulate in ES cell lines, and these may sometimes cause phenotypic effects independent of the knockout. Yet it is rare that an investigator actually maps a phenotype to the knockout locus to assure that cause and effect are being witnessed. And as already mentioned, some knockout mutations are simply lethal at an early embryonic stage and give little insight into the function of the gene during development or subsequently.

This depends upon the phenomenon under analysis, and how many genes are required for it to operate. The “genomic footprint” of a phenotype may be very large (millions of nucleotides) or very small (a few hundred nucleotides). In our laboratory, we study innate immunity, which depends upon thousands of genes. Hence, aberrant phenotypes are encountered very frequently. In addition, we encounter visible phenotypes (altered coat color, coat quality, morphology, metabolism, or behavior, for example) with some regularity.

All phenotypes tend to saturate at the same rate. Whether 10 genes or 1,000 genes are targets, 10%, 20%, and 30% saturation will be reached through screening of about the same number of mice. As a rule of thumb, about 10,000 G3 mice must be examined in order to identify 10% of the target genes. This assumes that G1 animals are bred to assure capture of about half of their mutations in homozygous form.

Genetics has been the single most successful branch of biology. All other disciplines in the life sciences depend upon it. While physicists and chemists explore questions of vast complexity, they do not have a tool that can quite be compared to genetics. The ocean may be complex. A star may be complex. A mountain may be complex. But unlike a human or a mouse, none of these inanimate objects provides a blueprint that tells how it is made. The genome of every living organism does just that.

In a practical sense, we know that genetics is important because humans suffer from genetic diseases: “simple” diseases like cystic fibrosis, sickle cell anemia, Tay Sach’s disease, and more “complex” diseases like juvenile diabetes. Cancer is ultimately a genetic disease. And paradoxically, so are some diseases that most would call infectious. Genetics is also important because it ultimately has the power to tell why we are just who and what we are. It tells us our place in the tree of life. What, after all, separates us from chimpanzees, and even from mice, but a definable set of mutations? Genetics even has the power to predict the future, to a moderate level of certainty. Who will live and who will die during the next decade? Genetics can begin to tell us.

But for those who are merely intellectually curious, genetics is important as well. It holds the power to tell us exactly how a living organism, be it a mouse or a worm or a tree or a human, actually fits together and works as the complex machine that it is.